Structural basis for feedback and pharmacological inhibition of Saccharomyces cerevisiae glutamate cysteine ligase

Ekaterina I. Biterova, Joseph J. Barycki

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of Saccharomyces cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione. Examination of the structure of ScGCL-glutathione complex (2.5 Å ; R = 19.9%, Rfree = 25.1%) indicates that the inhibitor occupies both the glutamate- and the presumed cysteine-binding site and disrupts the previously observed Mg2+ coordination in the ATP-binding site. L-Buthionine-S-sulfoximine (BSO) is a mechanism-based inhibitor of GCL and has been used extensively to deplete glutathione in cell culture and in vivo model systems. Inspection of the ScGCL-BSO structure (2.2 Å ; R = 18.1%, Rfree = 23.9%) confirms that BSO is phosphorylated on the sulfoximine nitrogen to generate the inhibitory species and reveals contacts that likely contribute to transition state stabilization. Overall, these structures advance our understanding of the molecular regulation of this critical enzyme and provide additional details of the catalytic mechanism of the enzyme.

Original languageEnglish (US)
Pages (from-to)14459-14466
Number of pages8
JournalJournal of Biological Chemistry
Volume285
Issue number19
DOIs
StatePublished - May 7 2010

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Glutamate-Cysteine Ligase
Yeast
Saccharomyces cerevisiae
Buthionine Sulfoximine
Glutathione
Pharmacology
Feedback
Enzymes
Binding Sites
Biosynthesis
Cell culture
Cysteine
Glutamic Acid
Nitrogen
Stabilization
Cell Culture Techniques
Adenosine Triphosphate
Inspection
Substrates
buthionine

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Structural basis for feedback and pharmacological inhibition of Saccharomyces cerevisiae glutamate cysteine ligase. / Biterova, Ekaterina I.; Barycki, Joseph J.

In: Journal of Biological Chemistry, Vol. 285, No. 19, 07.05.2010, p. 14459-14466.

Research output: Contribution to journalArticle

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